HIGH THROUGHPUT LIGHT SHEET MICROSCOPE WITH ADJUSTABLE ANGULAR ILLUMINATION
A light sheet microscope comprises a detection optics with a tilted focal plane, and an illumination optics generating a tilted light sheet. The light sheet may be rotated about a rotation axis in order to match the tilted focal plane. A multiple sample carrier translates multiple samples through the tilted light sheet in a translation direction which is not in the plane of the light sheet, thereby enabling acquisition of three-dimensional images of each of the multiple samples in a single pass through the light sheet.
The invention relates in general to high throughput imaging of samples with a light sheet microscope, and in particular to a light sheet microscope with adjustable angular illumination and angled detection for high throughput imaging of multiple samples.
BACKGROUND OF THE INVENTIONIn light sheet microscopes of existing practice, a substantially planar sheet of light enters a sample along a direction intersecting the detection light axis of a detection optical system. A three-dimensional image of the specimen is acquired by means of fluorescence radiation from the specimen that is detected by the detection optical system. Because no regions other than the image acquisition plane are irradiated with light, it is possible to acquire a superior three-dimensional image of the sample.
Today, this technique is gaining attention not only as a technique for obtaining a three-dimensional image of a living organism in which target molecules are labeled with fluorescent proteins, but also as a technique that is applied to drug development screening, in which pharmaceutical efficacy is evaluated by obtaining three-dimensional images of cultured cells and tissues, such as spheroids or organoids (artificial organs or portions thereof). When used for drug development screening, the technique is generally applied to a large number of samples, usually arrayed in a multi-sample carrier. In such cases, throughput of the sample analysis is a key parameter defining the efficiency and cost-effectiveness of the screening process. However, in light sheet microscopes of existing practice the throughput is limited by the need to make a separate fluorescent light acquisition for each imaging plane of the sample. Furthermore, light sheet configurations such as those disclosed by Siebenmorgen et al in US patent publications 2016/0154236 and 2017/0269345 propose structures which require illumination and detection objectives above or below the sample, the objectives being disposed at some angle to the (horizontal) sample translation direction. Such light sheet configurations may unnecessarily constrain the sample and be difficult to optimize optically. The solution proposed herein describes a simple structure which can achieve high throughput screening.
Brinkman and Shimada (European Patent Application EP3293559) have disclosed a multi-sample carrier for a light sheet microscope, the carrier comprising a rotating wheel or linear translation which enable samples to be rapidly translated horizontally through a horizontally oriented planar light sheet. Data is thereby acquired from a single plane of each of the samples. However, in order to obtain a full three-dimensional image of each sample, the horizontal stage translations must be stopped and a separate acquisition must be made for each desired imaging plane within the samples, with either the sample or the light sheet being moved in a vertical axis between successive imaging planes. Such sequential imaging is time consuming and limits the sample imaging throughput and therefore the cost-effectiveness of the image acquisition.
There therefore exists a need in existing practice for a higher throughput sample imaging light sheet microscope system having a simple optical structure.
SUMMARY OF THE INVENTIONAccordingly, it is a general objective of the present disclosure to have a high throughput sample imaging light sheet microscope.
This and other objectives are achieved by having a light sheet microscope which creates one or more light sheet illuminations that can be rotated around a rotation axis, together with an optical element that allows the detection objective to match a corresponding angled focal plane. Multiple samples are then swept through the sheet illumination by translating the samples in a translation direction, wherein the translation direction is not in the plane of the angled light sheet. In a preferred embodiment, the translation direction is perpendicular to the rotation axis and to the axis of the detection objective. In a single sweep of samples through the angled light sheet, a complete three-dimensional imaging data set is thereby generated for all the samples, allowing high throughput screening in three dimensions.
Note that for purposes of clear exposition, co-ordinates indicating X, Y, and Z directions are associated with
Note that cylindrical lens 6a is a preferred embodiment of light sheet rotation device 6 configured to form and rotate light sheet 8. However other optical devices may be used: for example, a spatial light modulator comprising electronic rotation of light sheet 8 may be used in place of mechanical rotation of cylindrical lens 6a. Generally, any suitable optical device or system may be used to form light sheet 8 and to rotate the plane of light sheet 8 about rotation axis 7, and all such optical devices or systems are within the scope of the present disclosure.
Continuing to refer to
In order to translationally align light sheet 8 with tilted focal plane 26 or with a second light sheet (see
In order to optimize the focus, detection optics 200 may optionally be placed in an assembly (not shown) that translates in a detection translation direction 15, which is preferably in the vertical Z direction. Alternatively, objective lens 14 may be fitted with an electronic focusing device (not shown) which may be adjusted to optimize the focus.
Any mechanisms or mechanical arrangements known in the art may be used for achieving motion in translation directions 5 or 15, and these mechanisms may be motorized and automated for convenience of adjustment.
Still referring to
Note that the structure of optical system 1 is much simpler than that of Siebenmorgen et al. which employs illumination and detection optics disposed at some angle from the sample translation direction. In contrast, optical system 1 comprises a detection optics which is vertical and perpendicular to a sample translation direction 29 (see
As shown above, it should be noted that one of the novel aspects of the invention is to provide at least one illumination optics producing at least one rotatable light sheet, and a detection optics providing a tilted focal plane. Another novel aspect is to provide alignment mechanisms so that the corresponding light sheet and tilted focal plane are aligned to be substantially coincident prior to conducting a measurement or a series of measurements.
Note that the foregoing embodiments of tilting device 12 are not intended to be limiting. Other optical devices capable of tilting the focal plane of objective lens 14 are possible, and all such devices are within the scope of the present disclosure.
It should be noted that, if the tilting angle is large (greater than about 30 degrees), the various embodiments of tilting device 12 described above may cause optical aberrations which might adversely affect the quality of the sample image. For example, prism 27 may cause chromatic aberrations of the image. The function of aberration correcting device 17 (see
Aberration correcting device 17 may comprise a single optical element or multiple optical elements. In an embodiment, aberration correcting device 17 comprises a single prism configured to primarily compensate chromatic aberrations introduced by tilting device 12. In another embodiment, aberration correcting device 17 comprises a liquid crystal on silicon (LCoS) device. A LCoS device is two-dimensional array of pixels, each pixel comprising a layer of liquid crystal material whose refractive index may be varied by application of voltage to the pixel. Spatially varying the refractive index of pixels over the surface of the LCoS array allows aberrations to be compensated in images transmitted through the array, the compensation being adjustable in real time.
Still referring to
Thus, by maximizing uniformity to achieve parallelism in step 52, and maximizing intensity to achieve coincidence in step 54, adjustment method 40 ensures co-planarity of light sheet 8 with tilted focal plane 26.
It should be noted that the “adjusting steps” associated with method 40 are preferably used as a periodic calibration procedure prior to testing one or more batches of samples. Once maximum uniformity and intensity of the light signal have been achieved, rotation of the illumination optics and other optical translations of method 40 are not required until the next calibration procedure.
Although the present invention has been described in relation to particular embodiments thereof, it can be appreciated that various designs can be conceived based on the teachings of the present disclosure, and all are within the scope of the present disclosure.
Claims
1. A light sheet microscope comprising:
- a set of detection optics configured to detect an emitted light from a sample, the detection optics comprising an objective lens having an optical axis and a normal focal plane perpendicular to the optical axis;
- a tilting device configured to tilt the normal focal plane of the detection optics such that a tilted focal plane is tilted with respect to the normal focal plane; and,
- an illumination optics generating at least one tilted light sheet, the illumination optics comprising at least one light sheet rotation device, wherein the light sheet rotation device is configured to rotate the light sheet about a rotation axis such that a light sheet plane of each of the at least one tilted light sheet is substantially coincident with the tilted focal plane.
2. The microscope of claim 1 further comprising a translating stage configured to move the sample through the at least one tilted light sheet in a sample translation direction, thereby generating the emitted light, and wherein the sample translation direction is not in the light sheet plane.
3. The microscope of claim 2 wherein the optical axis is vertical, the rotation axis is substantially perpendicular to the optical axis and the sample translation direction is substantially perpendicular to the rotation axis and the optical axis.
4. The microscope of claim 1 wherein the light sheet rotation device is a cylindrical lens configured to rotate about the rotation axis.
5. The microscope of claim 1 wherein the tilting device is a prism located between the objective lens and the sample.
6. The microscope of claim 1 wherein the tilting device is at least one mirror located between the objective lens and the sample.
7. The microscope of claim 1 wherein the tilting device is a gradient refractive index lens located between the objective lens and the sample.
8. The microscope of claim 1 wherein the tilting device is an electronically controlled gradient index device located between the objective lens and the sample.
9. The microscope of claim 2 wherein the translating stage carries multiple samples and wherein each of the multiple samples is sequentially translated through the at least one tilted light sheet in a single translation step of the translating stage.
10. The microscope of claim 9 further comprising an image acquisition system configured to acquire data to form a three-dimensional image of the emitted light from each of the multiple samples during the single translation step.
11. The microscope of claim 4 further comprising a light detecting device for measuring a total intensity and a uniformity of intensity of the emitted light in the tilted focal plane.
12. The microscope of claim 11 further comprising a rotation mechanism for rotating the cylindrical lens so as to maximize the uniformity of intensity.
13. The microscope of claim 11 further comprising an illumination optics translation mechanism for translating the illumination optics in a direction substantially parallel to the optical axis so as to maximize the total intensity.
14. The microscope of claim 11 further comprising a detection optics translation mechanism for translating the detection optics and the tilting device in a direction substantially parallel to the optical axis so as to maximize the total intensity.
15. The microscope of claim 11 wherein the detection optics comprises an electronically tunable lens and wherein the electronically tunable lens may be adjusted to maximize the total intensity.
16. The microscope of claim 1 wherein the detection optics further comprises an aberration correcting device configured to correct optical aberrations caused by the tilting device.
17. The microscope of claim 16 wherein the aberration correcting device is a prism.
18. The microscope of claim 16 wherein the aberration correcting device is a liquid crystal on silicon (LCoS) device.
19. A method of adjusting an optical system for a light sheet microscope, the method comprising the steps of:
- providing a detection optics comprising an objective lens having an optical axis and a normal focus plane perpendicular to the optical axis, the detection optics further comprising a light detecting device for measuring a total intensity and a uniformity of intensity of an emitted light from a sample;
- providing a tilting device configured to tilt a focal plane of the detection optics such that a tilted focal plane is tilted with respect to the normal focus plane;
- providing an illumination optics generating at least one tilted light sheet, each of the at least one tilted light sheet having a light sheet plane; and,
- rotating the light sheet plane to maximize the uniformity of intensity.
20. The method of claim 19, further comprising the step of translating the illumination optics in a direction substantially parallel to the optical axis so as to maximize the total intensity.
21. The method of claim 19, further comprising the step of translating the detection optics and the tilting device in a direction substantially parallel to the optical axis so as to maximize the total intensity.
22. The method of claim 19 wherein the detection optics comprises an electronic lens and the method further comprises the step of adjusting the electronic lens so as to maximize the total intensity.
23. The method of claim 19 wherein steps of the method represent steps for a calibration of the microscope.
24. A method of generating a three-dimensional image of an emitted light from each one of multiple samples with a light sheet microscope, the method comprising the steps of:
- providing a detection optics comprising an objective lens having a vertical optical axis and a normal focus plane perpendicular to the optical axis, the detection optics configured to detect the emitted light;
- providing a tilting device configured to tilt a focal plane of the detection optics such that a tilted focal plane is tilted with respect to the normal focus plane;
- providing an illumination optics generating at least one tilted light sheet, each of the at least one tilted light sheet having a light sheet plane;
- rotating the light sheet plane so that it is substantially coincident with the tilted focal plane;
- translating the multiple samples in a translation direction which is not in the light sheet plane, wherein the multiple samples are sequentially translated in a single translation step through the at least one tilted light sheet, thereby generating the emitted light; and,
- generating the three-dimensional image of each one of the multiple samples from the emitted light during the single translation step.
Type: Application
Filed: Sep 20, 2018
Publication Date: Mar 26, 2020
Inventor: Brendan Brinkman (Hopkinton, MA)
Application Number: 16/137,240